Expression of G protein coupled receptors in yeast

ABSTRACT

Disclosed is a transformed yeast cell containing a first heterologous DNA sequence which codes for a mammalian G protein-coupled receptor and a second heterologous DNA sequence which codes for a mammalian G protein α subunit (mammalian G α ). The first and second heterologous DNA sequences are capable of expression in the cell, but the cell is incapable of expressing an endogenous G protein α-subunit (yeast G α ). The cells are useful for screening compounds which affect the rate of dissociation of G α  from G βγ  in a cell. Also disclosed is a novel DNA expression vector useful for making cells as described above. The vector contains a first segment comprising at least a fragment of the extreme amino-terminal coding sequence of a yeast G protein-coupled receptor. A second segment is positioned downstream from the first segment (and in correct reading frame therewith), with the second segment comprising a DNA sequence encoding a heterologous G protein-coupled receptor.

This application is a divisional application of Ser. No. 09/752,145,filed Dec. 29, 2000 now U.S. Pat. No. 6,855,550, Issuing, which is adivisional application of Ser. No. 09/056,920, filed Apr. 8, 1998, nowU.S. Pat. No. 6,168,927, which in turn is a continuation application ofSer. No. 08/441,291, filed May 15, 1995, now U.S. Pat. No. 5,739,029,which is a divisional application of Ser. No. 08/071,355, filed Jun. 3,1993, now U.S. Pat. No. 5,482,835, which is a continuation applicationof Ser. No. 07/581,714, filed Sep. 13, 1990, now abandoned. Thedisclosures of all of the aforementioned applications/patents areincorporated herein in their entireties by reference.

This invention was made with government support under NIH grants HL16037and GM21841. The government may have certain rights to this invention.

FIELD OF THE INVENTION

This invention relates to yeast cells expressing heterologous G proteincoupled receptors, vectors useful for making such cells, and methods ofusing the same.

BACKGROUND OF THE INVENTION

The actions of many extracellular signals (for example,neurotransmitters, hormones, odorants, light) are mediated by receptorswith seven transmembrane domains (G protein coupled receptors) andheterotrimeric guanine nucleotide-binding regulatory proteins (Gproteins). See H. Dohlman, M. Caron, and R. Lefkowitz, Biochemistry 26,2657 (1987); L. Stryer and H. Bourne, Ann. Rev. Cell Biol. 2, 391(1988). Such G protein-mediated signaling systems have been identifiedin organisms as divergent as yeast and man. See H. Dohlman et al.,supra; L. Stryer and H. Bourne, supra; K. Blumer and J. Thorner, Annu.Rev, Physiol. (in press). The β2-adrenergic receptor (βAR) is theprototype of the seven-transmembrane-segment class of ligand bindingreceptors in mammalian cells. In response to epinephrine ornorepinephrine, βAR activates a C protein, G_(s), which in turnstimulates adenylate cyclase and cyclic adenosine monophosphateproduction in the cell. See H. Dohlman et al., supra; L. Stryer and H.Bourne, supra. G protein-coupled pheromone receptors in yeast control adevelopmental program that culminates in mating (fusion) of a and ahaploid cell types to form the a/α diploid. See K. Blumer and J.Thorner, supra; I. Herskowitz, Microbiol. Rev. 52, 536 (1988).

The present invention is based on our continued research into theexpression of heterologous G protein coupled receptors in yeast.

SUMMARY OF THE INVENTION

A first aspect of the present invention is a transformed yeast cellcontaining a first heterologous DNA sequence which codes for a mammalianG protein coupled receptor and a second heterologous DNA sequence whichcodes for a mammalian G protein α subunit (mammalian G_(α)). The firstand second heterologous DNA sequences are capable of expression in thecell, but the cell is incapable of expressing an endogenous G proteinα-subunit (yeast G_(α)). The cell optionally contains a thirdheterologous DNA sequence, with the third heterologous DNA sequencecomprising a pheromone-responsive promotor and an indicator genepositioned downstream from the pheromone-responsive promoter andoperatively associated therewith.

A second aspect of the present invention is a method of testing acompound for the ability to affect the rate of dissociation of G_(α)from G_(βγ) in a cell. The method comprises: providing a transformedyeast cell as described above; contacting the compound to the cell; andthen detecting the rate of dissociation of G_(α) from G_(βγ) in thecell. The cells may be provided in an aqueous solution, and thecontacting step carried out by adding the compound to the aqueoussolution.

A third aspect of the present invention is a DNA expression vectorcapable of expressing a transmembrane protein into the cell membrane ofyeast cells. The vector contains a first segment comprising at least afragment of the extreme amino-terminal coding sequence of a yeast Gprotein coupled receptor. A second segment is positioned downstream fromthe first segment (and in correct reading frame therewith), with thesecond segment comprising a DNA sequence encoding a heterologous Gprotein coupled receptor.

A fourth aspect of the present invention is a yeast cell transformed bya vector as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the construction of the yeast human β2 AdrenergicReceptor expression plasmid, pYβAR2, which includes nucleic acid (SEQ IDNO: 1) and the encoded amino acid sequences (SEQ ID NO: 2) of STE2.

FIG. 2 illustrates hβAR ligand binding to membranes frompYβAR2-transformed yeast cells.

FIG. 3 shows a comparison of β-adrenergic agonist effects onpheromone-inducible gene activity. α-MF, 10 μM α-mating factor; (−) ISO,50 μM (−) isoproterenol; (−) ALP, 50 μM (−) alprenolol; (+) ISO, 100 μM(+) isoproterenol.

DETAILED DESCRIPTION OF THE INVENTION

Nucleotide bases are abbreviated herein as follows:

A = Adenine C = Cytosine G = Guanine T = Thymine

Amino acid residues are abbreviated herein to either three letters or asingle letter as follows:

Ala; A = Alanine Arg; R = Arginine Asn; N = Asparagine Asp; D = Asparticacid Cys; C = Cysteine Gln; Q = Glutamine Glu; E = Glutamic acid Gly; G= Glycine His; H = Histidine Ile; I = Isoleucine Leu; L = Leucine Lys; K= Lysine Met; M = Methionine Phe; F = Phenylalanine Pro; P = ProlineSer; S = Serine Thr; T = Threonine Trp; W = Tryptophan Tyr; Y = TyrosineVal; V = Valine

The term “mammalian” as used herein refers to any mammalian species(e.g., human, mouse, rat, and monkey).

The term “heterologous” is used herein with respect to yeast, and hencerefers to DNA sequences, proteins, and other materials originating fromorganisms other than yeast (e.g., mammalian, avian, amphibian), orcombinations thereof not naturally found in yeast.

The terms “upstream” and “downstream” are used herein to refer to thedirection of transcription and translation, with a sequence beingtranscribed or translated prior to another sequence being referred to as“upstream” of the latter.

G proteins are comprised of three subunits: a guanyl-nucleotide bindingα subunit; a γ subunit; and a γ subunit. G proteins cycle between twoforms, depending on whether GDP or GTP is bound thereto. When GDP isbound the G protein exists as an inactive heterotrimer, the G_(αβγ)complex. When GTP is bound the α subunit dissociates, leaving a G_(βγ)complex. Importantly, when a G_(αβγ) complex operatively, associateswith an activated G protein coupled receptor in a cell membrane, therate of exchange of GTP for bound GDP is increased and, hence, the rateof dissociation of the bound α subunit from the G_(βγ) complexincreases. This fundamental scheme of events forms the basis for amultiplicity of different cell signaling phenomena. See generally Stryerand Bourne, supra.

Any mammalian G protein coupled receptor, and the DNA sequences encodingthese receptors, may be employed in practicing the present invention.Examples of such receptors include, but are not limited to, dopaminereceptors, muscarinic cholinergic receptors, α-adrenergic receptors,β-adrenergic receptors, opiate receptors, cannabinoid receptors, andserotonin receptors. The term receptor as used herein is intended toencompass subtypes of the named receptors, and mutants and homologsthereof, along with the DNA sequences encoding the same.

The human D₁ dopamine receptor cDNA is reported in A. Dearry et al.,Nature 347, 72-76 (1990).

The rat D₂ dopamine receptor cDNA is reported in J. Bunzow et al.,Nature 336, 783-787 (1988); see also O. Civelli, et al., PCT Appln. WO90/05780 (all references cited herein are to be incorporated herein byreference).

Muscarinic cholinergic receptors (various subtypes) are disclosed in E.Peralta et al., Nature 343, 434 (1988) and K. Fukuda et al., Nature 327,623 (1987).

Various subtypes of α₂-adrenergic receptors are disclosed in J. Regan etal., Proc. Natl. Acad. Sci. USA 85, 6301 (1988) and in R. Lefkowitz andM. Caron, J. Biol. Chem. 263, 4993 (1988).

Serotonin receptors (various subtypes) are disclosed in S. Peroutka,Ann. Rev. Neurosci. 11, 45 (1988).

A cannabinoid receptor is disclosed in L. Matsuda et al., Nature 346,561 (1990).

Any DNA sequence which codes for a mammalian G α subunit (G_(α)) may beused to practice the present invention. Examples of rammalian G αsubunits include G_(s) α subunits, G₁ α subunits, G_(o) α subunits,G_(z) α subunits, and transducin α subunits. See generally Stryer andBourne, supra. G proteins and subunits useful for practicing the presentinvention include subtypes, and mutants and homologs thereof, along withthe DNA sequences encoding the same.

Heterologous DNA sequences are expressed in a host by means of anexpression vector. An expression vector is a replicable DNA construct inwhich a DNA sequence encoding the heterologous DNA sequence is operablylinked to suitable control sequences capable of effecting the expressionof a protein or protein subunit coded for by the heterologous DNAsequence in the intended host. Generally, control sequences include atranscriptional promoter, an optional operator sequence to controltranscription, a sequence encoding suitable mRNA ribosomal bindingsites, and (optionally) sequences which control the termination oftranscription and translation.

Vectors useful for practicing the present invention include plasmids,viruses (including phage), and integratable DNA fragments (i.e.,fragments integratable into the host genome by homologousrecombination). The vector may replicate and function independently ofthe host genome, as in the case of a plasmid, or may integrate into thegenome itself, as in the case of an integratable DNA fragment. Suitablevectors will contain replicon and control sequences which are derivedfrom species compatible with the intended expression host. For example,a promoter operable in a host cell is one which binds the RNA polymeraseof that cell, and a ribosomal binding site operable in a host cell isone which binds the endogenous ribosomes of that cell.

DNA regions are operably associated when they are functionally relatedto each other. For example: a promoter is operably linked to a codingsequence if it controls the transcription of the sequence; a ribosomebinding site is operably linked to a coding sequence if it is positionedso as to permit translation. Generally, operably linked means contiguousand, in the case of leader sequences, contiguous and in reading phase.

Transformed host cells of the present invention are cells which havebeen transformed or transfected with the vectors constructed usingrecombinant DNA techniques and express the protein or protein subunitcoded for by the heterologous DNA sequences. In general, the host cellsare incapable of expressing an endogenous G protein α-subunit (yeastG_(α)). The host cells do, however, express a complex of the G protein βsubunit and the G protein γ subunit (G_(βγ)). The host cells may expressendogenous G_(βγ), or may optionally be engineered to expressheterologous G_(βγ) (e.g., mammalian) in the same manner as they areengineered to express heterologous G_(α).

A variety of yeast cultures, and suitable expression vectors fortransforming yeast cells, are known. See, e.g., U.S. Pat. No. 4,745,057;U.S. Pat. No. 4,797,359; U.S. Pat. No. 4,615,974; U.S. Pat. No.4,880,734; U.S. Pat. No. 4,711,844; and U.S. Pat. No. 4,865,989.Saccharomyces cerevisiae is the most commonly used among the yeast,although a number of other strains are commonly available. See, e.g.,U.S. Pat. No. 4,806,472 (Kluveromyces lactis and expression vectorstherefor); U.S. Pat. No. 4,855,231 (Pichia pastoris and expressionvectors therefor). Yeast vectors may contain an origin of replicationfrom the 2 micron yeast plasmid or an autonomously replicating sequence(ARS), a promoter, DNA encoding the heterologous DNA sequences,sequences for polyadenylation and transcription termination, and aselection gene. An exemplary plasmid is YRp7, (Stinchcomb et al., Nature282, 39 (1979); Kingsman et al., Gene 7, 141 (1979); Tschemper et al.,Gene 10, 157 (1980)). This plasmid contains the trpl gene, whichprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1(Jones, Genetics 85, 12 (1977)). The presence of the trpl lesion in theyeast host cell genome then provides an effective environment fordetecting transformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol.Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Req. 7, 149 (1968); and Holland et al., Biochemistry 17, 4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Suitable vectors and promoters for use in yeast expressionare further described in R. Hitzeman et al., EPO Publn. No. 73,657.Other promoters, which have the additional advantage of transcriptioncontrolled by growth conditions, are the promoter regions for alcoholdehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymesassociated with nitrogen metabolism, and the aforementionedmetallothionein and glyceraldehyde-3-phosphate dehydrogenase, as well asenzymes responsible for maltose and galactose utilization.

In constructing suitable expression plasmids, the termination sequencesassociated with these genes may also be ligated into the expressionvector 3′ of the heterologous coding sequences to providepolyadenylation and termination of the mRNA.

A novel DNA expression vector described herein which is particularlyuseful for carrying out the present invention contains a first segmentcomprising at least a fragment of the extreme amino-terminal codingsequence of a yeast G protein coupled receptor and a second segmentdownstream from said first segment and in correct reading frametherewith, the second segment comprising a DNA sequence encoding aheterologous G protein coupled receptor (e.g., a mammalian G proteincoupled receptor). In a preferred embodiment, this vector comprises aplasmid. In constructing such a vector, a fragment of the extremeamino-terminal coding sequence of the heterologous G protein coupledreceptor may be deleted. The first and second segments are operativelyassociated with a promoter, such as the GAL1 promoter, which isoperative in a yeast cell. Coding sequences for yeast G protein coupledreceptors which may be used in constructing such vectors are exemplifiedby the gene sequences encoding yeast phereomone receptors (e.g., theSTE2 gene, which encodes the α-factor receptor, and the STE3 gene, whichencodes the a-factor receptor). The levels of expression obtained fromthese novel vectors are enhanced if at least a fragment of the5′-untranslated region of a yeast G protein coupled receptor gene (e.g.,a yeast pheromone receptor gene; see above) is positioned upstream fromthe first segment and operatively associated therewith.

Any of a variety of means for detecting the dissociation of G_(α) fromG_(βγ) can be used in connection with the present invention. The cellscould be disrupted and the proportion of these subunits and complexesdetermined physically (i.e., by chromatography). The cells could bedisrupted and the quantity of G alpha present assayed directly byassaying for the enzymatic activity possessed by G_(α) in isolation(i.e., the ability to hydrolyze GTP to GDP). Since whether GTP or GDP isbound to the G protein depends on whether the G protein exists as aG_(βγ) or G_(αβγ) complex, dissociation can be probed with radiolabelledGTP. As explained below, morphological changes in the cells can beobserved. A particularly convenient method, however, is to provide inthe cell a third heterologous DNA sequence, wherein the thirdheterologous DNA sequence comprises a pheromone-responsive promotor andan indicator gene positioned downstream from the pheromone-responsivepromoter and operatively associated therewith. This sequence can beinserted with a vector, as described in detail herein. With such asequence in place, the detecting step can be carried out by monitoringthe expression of the indicator gene in the cell. Any of a variety ofpheromone responsive promoters could be used, examples being the BAR1gene promoter and the FUS1gene promoter. Likewise, any of a broadvariety of indicator genes could be used with examples including theHIS3 gene and the LacZ gene.

As noted above, transformed host cells of the present invention expressthe protein or protein subunit coded for by the heterologous DNAsequence. When expressed, the G protein coupled receptor is located inthe host cell membrane (i.e., physically positioned therein in properorientation for both the stereospecific binding of ligands on theextracellular side of the cell membrane and for functional interactionwith G proteins on the cytoplasmic side of the cell membrane).

The ability to control the yeast pheromone response pathway byexpression of a heterologous adrenergic receptor and its cognate Gprotein α-subunit has the potential to facilitate structural andfunctional characterization of mammalian G protein-coupled receptors. Byscoring for responses such as growth arrest or β-galactosidaseinduction, the functional properties of mutant receptors can now berapidly tested. Similarly, as additional genes for putative Gprotein-coupled receptors are isolated, numerous ligands can be screenedto identify those with activity toward previously unidentifiedreceptors. See F. Libert et al., Science 244, 569 (1989); M . S. Chee etal., Nature 344, 774 (1990). Moreover, as additional genes coding forputative G protein α-subunits are isolated, they can be expressed incells of the present invention and screened with a variety of G proteincoupled receptors and ligands to characterize these subunits. Thesecells can also be used to screen for compounds which affect receptor-Gprotein interactions.

Cells of the present invention can be deposited in the wells ofmicrotiter plates in known, predetermined quantities to providestandardized kits useful for screening compounds in accordance with thevarious screening procedures described above.

The following Examples are provided to further illustrate variousaspects of the present invention. They are not to be construed aslimiting the invention.

EXAMPLE 1 Construction of the Human β2-Adrenergic Expression VectorpYβAR2 and Expression in Yeast

To attain high level expression of the human β2-adrenergic receptor(hβAR) in yeast, a modified hβAR gene was placed under the control ofthe GAL1 promoter in the multicopy vector, YEp24 (pYβAR2).

FIG. 1 illustrates the construction of yeast expression plasmid pYβAR2.In pYβAR2, expression of the hβAR sequence is under the control of theGAL1 promoter. FIG. 1A shows the 5′-untranslated region and the first 63basepairs (bp) of coding sequence of the hβAR gene in pTZNAR, B. O'Dowdet al., J. biol. Chem. 263, 15985 (1988), which was removed by Aat IIcleavage and replaced with a synthetic oligonucleotide corresponding to11 bp of noncoding and 42 bp of coding sequence from the STE.2 gene. SeeN. Nakayama et al., EMBO J. 4, 2643 (1985); A. Burkholder and L.Hartwell, Nucleic Acids Res. 13, 8463 (1985). The resulting plasmid,pTZYNAR, contains the modified hβAR gene flanked by Hind III sites innoncoding sequences. The Hind III-Hind III fragment was isolated frompTZYNAR and inserted into pAAH5 such that the 3′-untranslated sequenceof the modified hβAR gene was followed by 450 bp containing terminationsequences from the yeast ADH1 gene. See G. Ammerer, Methods. Enzymol.101, 192 (1983).

As illustrated in FIG. 1B, pyβ13AR2 was constructed by inserting the BamHI-Bam HI fragment containing hβAR and ADJ1 sequences into YEpG24. E.Wyckoff and T. Hsieh, Proc. Natl. Acad. Sci. U.S.A. 85, 6272 (1988).Where maximum expression was sought, cells were cotransformed withplasmid pMTL9 (from Dr. S. Johnston) containing LAC9, a homolog of theS. cerevisiae GAL4 transactivator protein required for GAL1-regulatedtranscription. J. Salmeron et al., Mol. Cell. Biol. 9, 2950 (1989).Cells grown to late exponential phase were induced in medium containing3% galactose, supplemented with about 10 μM alprenolol, and grown for anadditional 36 hours. Standard methods for the maintenance of cells wereused. See F. Sherman et al., Methods in Yeast Genetics (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1986).

Maximal expression required (i) expression of a transcriptionaltransactivator protein (LAC9), (ii) replacement of the 5′ untranslatedand extreme NH₂-terminal coding sequence of the hβAR gene with thecorresponding region of the yeast STE2 (α-factor receptor) gene, (iii)induction with galactose when cell growth reached late exponentialphase, and, (iv) inclusion of an adrenergic ligand in the growth mediumduring induction.

The plasmid pYβAR2 was deposited in accordance with the provisions ofthe Budapest Treaty at the American Type Culture Collection, 12301Parklawn Drive, Rockville, Md. 20852 USA, on Sep. 11, 1990, and has beenassigned ATCC Accession No. 40891.

EXAMPLE 2 Binding Affinity of hβAR Ligands in Yeast Transformed withpYβAR2

A primary function of cell surface receptors is to recognize onlyappropriate ligands among other extracellular stimuli. Accordingly,ligand binding affinities were determined to establish the functionalintegrity of hβAR expressed in yeast. As discussed in detail below, anantagonist, ¹²⁵I-labeled cyanopindolol (¹²⁵I-CYP), bound in a saturablemanner and with high affinity to membranes prepared frompYβAR2-transformed yeast cells. By displacement of ¹²⁵I-CYP with aseries of agonists, the order of potency and stereospecificity expectedfor hβAR was observed.

SC261 cells (MATa ura3-52 trpl leu2 prbl-1122 pep4-3 prcl-407) (from Dr.S. Johnston) harboring pYβAR2 (URA3) and pMTL9 (LEU2) were grown inminimal glucose-free selective media to late log phase (OD₆₀₀=5.0), andthen induced with the addition of 3% galactose and 40 μM alprenolol.After 36 hours, cells were harvested and spheroplasts were prepared asdescribed. See E. Wyckoff and T. Hsieh, Proc. Natl. Acad. Sci. U.S.A.85, 6272 (1988). Briefly, the spheroplasts were resuspended in 50 mMTris-HCl pH 7.4, 5 mM EDTA and were lysed by vortex mixing with glassbeads for three one-min periods at 4° C. Crude membranes were preparedfrom the lysates and binding assays with ¹²⁵I-CYP were performed bymethods described previously. See H. Dohlman et al., Biochemistry 29,2335 (1990).

FIG. 2 illustrates hβAR ligand binding to membranes frompYβAR2-transformed yeast cells. (A) B_(max) (maximum ligand bound) andK_(d) (ligand dissociation constant) values were determined by varying125_(I)-CYP concentrations (5-400 pM). Specific binding was defined asthe amount of total binding (circles) minus nonspecific binding measuredin the presence of 10 μM (−) alprenolol (squares). A K_(d) of 93 pM for125_(I)-CYP binding was obtained and used to calculate agonistaffinities (below). (B) Displacement of 18 pM 125_(I)-CYP with variousconcentrations of agonists was used to determine apparent low affinityK_(t) values (non G protein coupled, determined in the presence of 50 μMGTP) for receptor binding, squares; (−) isoproterenol, circles; (−)epinephrine, downward-pointing triangles; (+) isoproterenol, upwardpointing triangles; (−) norepinephrine.

COMPARATIVE EXAMPLE A Ligand Binding Affinity for hβAR Expressed inYeast and Mammalian Cells

The binding data of FIGS. 2(A) and (B) were analyzed by nonlinear leastsquares regression, see A. DeLean et al., Mol. Pharmacol. 21, (1982),and are presented in Table I. Values given are averages of measurementsin triplicate, and are representative of 2-3 experiments. Bindingaffinities in yeast were nearly identical to those observed previouslyfor hβAR expressed in mammalian cells.

TABLE 1 Comparison of ligand Binding Parameters for High LevelExpression of Human β-Adrenergic Receptor in Yeast and COS-7 Cells*Yeast Monkey SC261 COS-7 (pY, βAR2, pMTL9) (pBC12: β, BAR) 125₁-CYP:¹K_(d) 0.093 nM ± 0.013 0.110 nM ± 0.009 ²B_(max) 115 pmol/mg 24 pmol/mg³K_(i)(M): (−) isoproterenol  103 ± 26  130 ± 15 (+) isoproterenol 3670± 420 4000 ± 184 (−) epinephrine  664 ± 123  360 ± 30 (−) norepinephrine6000 ± 1383 5800 ± 373 *Values derived from FIG. 2 and H. Dohlman etal., Biochemistry 29, 2335 (1990).; ± S.E. ¹K_(d), ligand dissociationconstant ²B_(max), maximum ligand bound ³K_(i), inhibition constant

EXAMPLE 3 Agonist-Dependent Activation of Mating Signal Transduction inYeast Expressing hβAR

A second major function of a receptor is agonist-dependent regulation ofdownstream components in the signal transduction pathway. Because thepheromone-responsive effector in yeast is not known, indirect biologicalassays are the most useful indicators of receptor functionality. See K.Blumer and J. Thorner, Annu. Rev. Physiol. in press; I. Herskowitz,Microbiol. Rev. 52, 536 (1988). In yeast cells expressing highconcentrations of hβAR, no agonist-dependent activation of the matingsignal transduction pathway could be detected by any of the typical invivo assays; for example, imposition of G1 arrest, induction of geneexpression alteration of morphology (so-called “shmoo” formation) orstimulation of mating. A likely explanation for the absence ofresponsiveness is that hβAR was unable to couple with the endogenousyeast G protein.

EXAMPLE 4 Coexpression of hβAR and Mammalian G_(s) α-Subunit in Yeast

Expression of a mammalian G_(s) α-subunit can correct the growth defectin yeast cells lacking the corresponding endogenous protein encoded bythe GPA1 gene. See C. Dietzel and J. Kurjan, Cell 50, 1001 (1987).Moreover, specificity of receptor coupling in mammalian cells isconferred by the α-subunit of G proteins. See L. Stryer and H. Bourne,Annu. Rev. Cell Biol. 2, 391 (1988). Thus, coexpression of hβAR and amammalian G_(s) α-subunit (GSα) in yeast was attempted to render theyeast responsive to adrenergic ligands. Accordingly, a cDNA encoding ratG_(s)α under the control of the copper-inducible CUP1 promoter wasintroduced on a second plasmid, pYSK136Gαs. See C. Dietzel and J.Kurjan, Cell 50, 1001 (1987). In yeast (NNY19) coexpressing hβAR and ratG_(s)α, but containing wild-type GPA1, no adrenergic agonist-inducedshmoo formation, a characteristic morphological change of yeast inresponse to mating pheromone, was observed.

EXAMPLE 5 Coexpression of hβAR and Mammalian G_(s) α-Subunit in YeastLacking an Endogenous G Protein α-Subunit

To prevent interference by the endogenous yeast G protein α-subunit,gpal mutant cells (strain 8c) were used.

Yeast strain 8c (MATa ura3 leu2 his3 trpl gpal::H153), I. Miyajima etal., Cell 50, 1011 (1987), carrying plasmids pYSK136Gαs (TRPl), C.Dietzel and J. Kurjan, Cell 50, 1001 (1987), pMTL9 (LEU2), J. Salmeronet al., Mol. Cell. Biol. 9, 2950 (1989), and pYβAR2 (URA3) wasmaintained on glucose-free minimal selective plates containing 3%glycerol, 2% lactic acid, 50 μM CUSO₄ and 3% galactose. Colonies weretransferred to similar plates containing 0.5 mM ascorbic acid and theindicated adrenergic ligand(s). After 16-20 hours at 30° C., thecolonies were transferred to similar liquid media at a density of10⁶-10⁷ cells/ml and examined by phase contrast microscopy.

Morphologies of yeast cells cotransformed with pYβAR2, pMTL9, andpYSK136Gαs were examined after incubation with (A) no adrenergic agent;(B) 100 μM (−) isoproterenol; (C) 100 μM (−) isoproterenol and 50 μM (−)alprenolol; and (D) 100 μM (+) isoproterenol. Results showed thattreatment of 8c cells coexpressing hβAR and rat G_(s)α with theβ-adrenergic agonist isoproterenol indeed induced shmoo formation, andthat this effect was blocked by the specific antagonist alprenolol.

EXAMPLE 6 Coexpression of hβAR and Mammalian G_(s)α-Subunit in YeastContaining a β-Galactosidase Signal Sequence

The isoproterenol-induced morphological response of 8c cellscoexpressing hβAR and rat G_(s)α suggested that these components cancouple to each other and to downstream components of the pheromoneresponse pathway in yeast lacking the endogenous G α-subunit. To confirmthat the pheromone signaling pathway was activated by hβAR and ratG_(s)α, agonist induction of the pheromone-responsive FUSl gene promoterwas measured in a strain of yeast derived from 8c cells (8cl) in which aFUS1-lacZ gene fusion had been stably integrated into the genome. See S.Nomoto et al., EMBO J. 9, 691 (1990).

Strains 8c (FIG. 3, legend) and NNY19 (MATa ura3 leu2 his3 trp1 lys2FUSl-LacZ::LEU2) were modified by integrative transformation withYIpFUS102 (LEU2), S. Nomoto et al., supra, and designated 8cl and NNY19,respectively. These strains were transformed with pYβAR2 and pYSK136Gαsand maintained on minimal selective plates containing glucose and 50 μMCuSO₄. Colonies were inoculated into minimal selective media (3%glycerol, 2% lactic acid, 50 μM CuSO₄), grown to early log phase(OD₆₀₀=1.0), and induced for 12 hours by addition of 3% galactose. Cellswere washed and resuspended in induction media (OD₆₀₀=5.0) containing0.5 mM ascorbic acid and the indicated ligands. After a 4 hourincubation at 30° C., cells were harvested, resuspended into 1 ml ofZ-buffer, see J. Miller, Experiments in Molecular Genetics (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1972), supplemented with0.0075% SDS, and β-galactosidase activities were determined in 3-4independent experiments as described previously. See J. Miller, supra.

FIG. 3 shows a comparison of β-adrenergic agonist effects onpheromone-inducible gene activity. α-MF, 10 μM α-mating factor; (−) ISO,50 μM (−) isoproterenol; (−) ALP, 50 μM (−) alprenolol; (+) ISO, 100 μM(+) isoproterenol. In 8cl (gpal) cells coexpressing hβAR and rat G_(s)α,a dramatic isoproterenol-stimulated induction of β-galactosidaseactivity was observed. Agonist stimulation was stereoselective and wasblocked by addition of a specific antagonist. Agonist responsiveness wasdependent on expression of both hβAR and rat G_(s)α, and required astrain in which the endogenous G protein α-subunit was disrupted. Thefinal β-galactosidase activity achieved in response to isoproterenol intransformed 8cl cells was comparable to that induced by α-factor innontransformed cells that express GPA1 (NNY19), although basalβ-galactosidase activity in NNY19 cells was considerably lower than in8cl cells. Taken together, our results indicated that coexpression ofhβAR and rat G_(s)α was sufficient to place under catecholamine controlkey aspects of the mating signal transduction pathway in yeast. However,the adrenergic agonist did not stimulate mating in either 8c cells orNNY19 cells coexpressing hβAR and rat G_(s)α, in agreement with recentobservations that yeast pheromone receptors, in addition to bindingpheromones, participate in other recognition events required for mating.See A. Bender and G. Sprague, Genetics 121, 463 (1989).

hβAR stimulates adenylate cyclase in animal cells via the action of theα-subunit of its G protein. In contrast, mating factor receptors inyeast trigger their effector via the action of the βγ subunits. M.Whiteway et al., Cell 56, 476 (1989). Our present results indicate thatactivation of hβAR in yeast leads to dissociation of mammalianG_(sα)from yeast βγ, and it is the βγ subunits that presumably elicitthe response.

The foregoing examples are illustrative of the present invention, andare not to be construed as limiting thereof. The invention is defined bythe following claims, with equivalents of the claims to be includedtherein.

1. A transformed yeast cell containing a first heterologous DNA sequencewhich codes for a mammalian G protein-coupled receptor and a secondheterologous DNA sequence which codes for a mammalian G protein αsubunit (mammalian G_(α)), wherein said first and second heterologousDNA sequences are capable of expression in said cell such that saidheterologous mammalian G protein-coupled receptor and said heterologousmammalian G protein α subunit can operatively associate, and whereinsaid cell is incapable of expressing an endogenous G protein α-subunit(yeast G_(α)).
 2. A transformed yeast cell according to claim 1, whereinsaid first heterologous DNA sequence is carried by a plasmid.
 3. Atransformed yeast cell according to claim 1, wherein said secondheterologous DNA sequence is carried by a plasmid.
 4. A transformedyeast cell according to claim 1, wherein said mammalian G protein αsubunit is selected from the group consisting of G_(S) α subunits, G_(L)α subunits, G_(O) α subunits, G_(Z) α subunits, and transducin αsubunits.
 5. A transformed yeast cell according to claim 1 whichexpresses a complex of the G protein β subunit and the G protein γsubunit (G_(βγ)).
 6. A transformed yeast cell according to claim 5 whichexpresses endogenous G_(βγ).
 7. A transformed yeast cell according toclaim 1, wherein said first heterologous DNA sequence codes for amammalian G protein-coupled receptor selected from the group consistingof dopamine receptors, muscarinic cholinergic receptors, α-adrenergicreceptors, β-adrenergic receptors, opiate receptors, cannabinoidreceptors, and scrotonin receptors.
 8. A transformed yeast cellaccording to claim 1 further comprising a third heterologous DNAsequence, wherein said third heterologous DNA sequence comprises apheromone-responsive promotor and an indicator gene positioneddownstream from said pheromone-responsive promoter and operativelyassociated therewith.
 9. A transformed yeast cell according to claim 8,wherein said pheromone responsive promoter is selected from the groupconsisting of the BAR1 gene promoter and the FUS1 gene promoter, andwherein said indicator gene is selected from the group consisting of theHIS3 gene and the LacZ gene.
 10. The transformed yeast cell according toclaim 1, wherein said mammalian G protein-coupled receptor and saidmammalian G protein α subunit operatively associate and activate anendogenous yeast signal transduction pathway.
 11. The transformed yeastcell according to claim 10, wherein said endogenous yeast signaltransduction pathway is a yeast pheromone response pathway.
 12. Theyeast cell of claim 11, further comprising a heterologous DNA sequenceincluding an indicator gene which is operatively associated with apheromone responsive promoter.
 13. The yeast cell of claim 12, whereinthe pheromone responsive promoter is selected from the group consistingof a BAR1 gene promoter and a Fus1 gene promoter.